In this document, researchers demonstrate that cyanobacteriochrome (CBCR) proteins found in spirulina are a viable natural alternative to artificial food dyes. They discovered novel CBCR proteins with varying light absorption spectra through metagenomic mining. Three candidate proteins expressed in E. coli matched predictions about their color. The researchers aim to adapt this work for expressing CBCRs in Bacillus subtilis to make them suitable for food applications.

1. Cyanobacteriochromes as a Viable Natural
Alternative to Synthetic Food Dyes
Jonathan Chen, Amanda S. Everitt, Samir Akre, Brent W. Weyers, Alexis A. Caligiuri
Motivation
Color is innate in food perception and consumers expect
vivid colors -- beyond those already naturally present in
food. However, due to backlash against artificial
colorants, some large food companies have pledged to
exclusively use natural food colorings.
Acknowledgements
Special thanks to our advisors for the countless hours of work they spent advising
us to ensure that our team continued progressing. Thanks to Nathan Rockwell and
Clark Lagarias of the Lagarias Lab at UC Davis for assisting us with their expertise
in CBCRs and Youtian Cui, Wai Shun Mak and Navneet Rai with their guidance.
Advisors: Marc T. Facciotti, Justin Siegel, Colin Milburn, Andrew Yao, Matthias Hess
Consumer Acceptance of Product
Identifies patterns in CBCR protein sequences based on their
intrinsic GAF domain; this model provides users with predictions on
what color the CBCR pigmented protein is likely to reflect. Try it out
at ucdigem.pythonanywhere.com
Collaboration Forum: iGEMmatch
iGEMmatch.org promotes collaborations between iGEM
teams. iGEM Match enables users to identify other teams
with compatible projects and provides a communication
modality to facilitate team-team collaboration.
Characterization of Novel GAF Proteins
Protein Production Optimization
Finding New Colors
To expand the color spectrum of natural food dyes, we
used mined metagenomic data to find novel CBCR
proteins with varying absorption spectra. We developed
a program that can predict whether the CBCRs will be
primarily blue, red, or yellow based on their sequence.
Metagenomic mining was used to discover 13 protein sequences three were transformed into E.coli and
subsequent spectrophotometric analysis were performed to find that our model correctly predicted all three GAF
sequences would reflect above 600 nm. The spectra shown here as compared to Blue #1.
We induced cultures with different concentrations of arabinose, and after harvesting we assessed pellet volume
and color vibrancy to see if there was an optimal inducer concentration.
We propose incremental and exponential movements toward a wealth of labels that allow accurate information
to consumers and open a realistic dialogue about the current human food system.
We created a guide to be used
as a tool for future iGEM
teams conducting surveys to
ensure that the data produced
does not violate human
experimentation rights and
can be used in future research
Enzymatic pathway for creation of
the bilin cofactor which binds to the
GAF domain of a CBCR giving it
color
Natural substitutes
for blue dye #1 are
thought to be the
hardest to find.
In this project we demonstrate that
cyanobacteriochrome (CBCR) proteins – like those
found in spirulina - are a viable natural alternative to
artificial food dyes.
Work in progress
Genetic Circuit for CBCR Expression in B. Subtilis
B. Subtilis KO7 (BGSC Accession # 1A1134): Seven protease
deletions, no major secreted proteases, free of antibiotic resistance
genes and integrated plasmids
B. Subtilis PY79 (BGSC Accession # 1A747): Wildtype strain,
containing normal secreted and wall-associated proteases
B. Subtilis spo0A3 (BGSC Accession # 1S1): Does not produce
alkaline or neutral proteases, non-sporulating
In order to use our CBCRs for food applications and
industrial production, our next step is to adapt our research
in E. coli for expression in Bacillus subtilis, which has been
designated by the FDA as a generally regarded as safe
(GRAS) organism.
This circuit architecture allows for the high-level constitutive production of
PCB cofactor (pVEG) and the independent tuning of of CBCR synthesis.
Expression Chassis to Explore
Can you tell
which is Blue
#1?
Would you buy genetically modified
foods if they were labeled?
Which would you prefer? Dye from
an altered organism, chemical
synthesis, or no preference?
Our software
was able to
connect more
than 55
international
teams.